Liposomes are central to drug delivery and membrane biology research. SPR provides a powerful label-free method for characterizing liposome binding and membrane interactions.
Introduction
Liposomes are phospholipid vesicles composed of concentric phospholipid bilayers that enclose to create an aqueous space. The phospholipid tails are made of two hydrophobic long fatty acid chains that aggregate together to minimize interactions with water molecules. Liposomes are of important interest in the field of drug delivery because they are non-toxic, biocompatible, and biodegradable. They can deliver drugs by having them inserted into the hydrophobic part of the bilayer or aqueous interior of the liposomes. In addition, liposomes can protect drugs from being degraded by enzymes and filtered out by the kidneys.
To deliver liposomes to a specific type of cell, liposomes are modified with an antibody, ligand, or other proteins or peptides. Once the modified liposomes are recognized by the specific receptors on target cells, they are taken up by the cell in a process called receptor-mediated endocytosis. For example, folate (a ligand) and its binding receptor, folate binding protein (FBP), are often used to study drug delivery strategies in cancer research because the FBP is over-expressed in a broad range of human cancers.
Poly(ethylene glycol) (PEG) is commonly used to prevent surface adsorption of proteins. For liposome applications, PEG is often added to extend the circulation time of liposomes in the human body because without it, the unmodified liposomes would be removed within a few hours by phagocytes. However, PEG may decrease the ability of the ligand on the liposome to bind to its specific receptor due to steric hindrance.
Experimental Procedures
The SPR measurements were performed using Affinité's P4SPR under static conditions. To immobilize the folate receptor, the 16-MHA-coated gold prism was activated through the injection of EDC:NHS 20:5 mM in MilliQ water and reacted for 20 minutes. Then, 40 nM folate binding protein (FBP) was injected and reacted for 1 h. Following FBP immobilization, 1 M ethanolamine was injected to deactivate unreacted NHS groups. Liposome samples at a concentration of 10 µM were injected and allowed to immobilize for 10 minutes. Between injections, the surface was rinsed sequentially with PBS and 10 mM glycine (pH 1.5).
Results
The data show that the P4SPR was capable of detecting liposomes via the folate and FBP binding affinity. In addition, one can see that the PEGylated, folate-modified liposome sample had a lower relative change in resonance units (~40 RU) compared to the unPEGylated folate-modified liposome sample (~110 RU). This demonstrates that the presence of PEG on the liposome surface inhibited some binding of the folate-modified liposomes to the FBP-modified sensor surface due to steric hindrance.
This experiment took about 3 hours to complete, from surface activation and folate modification of the sensor surface to the final injections of the liposome samples. The fact that a researcher can change experimental conditions at any moment is an advantage of having an SPR instrument such as the P4SPR in one's laboratory.
Conclusions
Affinité's P4SPR was able to detect folate-modified liposomes and more importantly, distinguish the differences between PEGylated and unPEGylated folate-modified liposomes due to the steric hindrance caused by PEG. Affinité's P4SPR can be used as a standard platform for researchers to characterize liposomes in a quick and simple manner prior to being further tested in bioassays or even in animals to obtain pharmacokinetic profiles. Having the P4SPR within reach would accelerate the research development of liposomes because there would not be a need to access a centralized SPR instrument.